Gold Salts
Robert Koch is credited with discovering the bacteriostatic effect of gold cyanide on Mycobacterium tuberculosis. It was subsequently observed that patients with tuberculosis often benefited from a reduction in certain inflammatory conditions when given gold salt injections for the disease. This observed reduction in inflammation led to aurothiolates being used by Forestier in 1927 as a treatment for rheumatoid arthritis (Panyala, 2009) (Abraham, 1997). The early gold-based products were typically injected in an intramuscular, or subcutaneous manner (and later in an intraarterial manner) and some are still available today and/or still being used to treat rheumatoid arthritis.
Specifically, it has been known for many years that certain gold compounds possess anti-inflammatory activity. For example, (i) sodium gold thiomalate (also referred to as “gold sodium thiomalate”), marketed as Myocrisin and related chemical versions, marketed as Myochrisine and Myochrisis; (ii) sodium gold thioglucose (also referred to as “gold sodium thioglucose”), marketed as Solganol; (iii) sodium gold thiosulfate, marketed as Sanocrysin and related chemical versions, marketed as Crisalbine, Aurothion and Sanocrysis; and (iv) sodium gold thiopropanolsulfonate, marketed as Allocrysine, have been used in the treatment of rheumatoid arthritis (Sadler, 1976; Shaw, 1999; Eisler, p. 133, 2004). Only monovalent gold salts were believed to exhibit therapeutic effects for the treatment of rheumatoid arthritis. In 1961 the Empire Rheumatism Council affirmed that injectable gold salts showed efficacy and gold salts remain a widely used method of treatment of progressive rheumatoid arthritis (Ueda, 1998).
Treatment with various gold salts has also been suggested, or anecdotally observed, to be effective in a range of other diseases, including asthma, HIV, malaria and cancer. A considerable body of evidence exists in these diseases, in both human and animal models, suggesting that gold may be a viable treatment option for these areas of unmet medical need (Dabrowiak, 2009).
Oral Gold
More recently, an oral gold product, 2,3,4,6-Tetra-o-acetyl I-thio B-D-gIucopyranosato-S-(triethyl-phosphine), marketed as Auranofin® or Ridaura® in several parts of the world, has become available (Ho & Tiekink, 2005, Dabrowiak, 2009). Auranofin® was approved by the FDA for human use in the mid-1980's; and Auranofin® had the advantage of being orally absorbed, but was considered to be less effective than the injectable gold thiolates (Sadler, 1976; Shaw 1999).
Toxicology of Gold Salts and Oral Gold
Historically, toxicity has limited the use of all injectable and oral gold-based therapies, with anywhere from 30-50% of patients terminating various gold-based treatments due to undesirable or intolerable side effects. The side effects of many conventional gold therapies include rashes or mucocutaneous effects (e.g., pruritus, dermatitis and stomatitis); hematologic changes (e.g., thrombocytopenia); protein in the urine (proteinuria); inflammation of the mouth; reduction in the number of circulating leukocytes; decreased number of blood platelets; aplastic anemia due to organ damage; lung abnormalities; adverse immune reactions, such as eosinophilia, lymphadenopathy, hypergamma globulinemia; severe hypotension, angina, myocardial infarction, nephrotoxicity and nephrotic syndrome; hepatitis; colitis; and chrysiasis (pigmentation) of the cornea, lens, and skin (Eisler, p. 133-134, 2004). The most common side effect of chrysotherapy was skin toxicity, accounting for up to 60% of all adverse reactions, especially lichenoid eruptions and non-specific dermatitis (Eisler, p. 133-134, 2004). These side effects are believed to be related to the formulations used (e.g., carrier molecule, oxidation state of the gold in the compound, etc.), rather than the gold itself (Ho & Tiekink, 2005).
Payne and Arena in 1978 reported the subacute and chronic toxicity of several oral gold compounds, including Auranofin®, in rats, compared to an injected gold control. Sprague Dawley rats were dosed for periods of 6 weeks, 6 months and one year. In a follow-up study, the 1-year investigation was repeated with sequential kills and a modified dosing regimen.
The target organs identified by this study were the stomach and kidney. Gastric changes consisted of superficial erosions of the mucosa extending up to ⅓ of the thickness of the mucosa and covering up to 5% of its surface area. This change was dose-related and was associated with loss of body weight. Healing lesions were also evident. In the kidney of rats given SK&F 36914 for six months there was enlargement of cortical tubular epithelial cells (cytomegaly). In addition, there was a dose-related enlargement of the nucleus (karyomegaly), with evidence of pleomorphic and multinucleate cells. In the 1-year study similar changes were seen, but in addition renal cortical cell adenomas were seen in a dose-related incidence (0/38, 3/39, 6/37 and 8/37 for control, low, intermediate and high dose respectively). In a repeated 1-year study an unexpectedly high incidence of mortality occurred. This was attributed to ileocaecal lesions that progressed to ulceration that appeared to perforate the gut wall in a number of cases. Presumably death resulted from acute infectious peritonitis. In the injected controls, gold sodium thiomalate was administered by intra-muscular injection once weekly for a year and, in a second study, once weekly for 46 weeks and then daily for 330 days. In the 1-year study, renal tubular cell karyomegaly was observed and renal cell adenoma was seen in 1/16 females but not in males. In the 21 month study all surviving rats showed karyomegaly of the renal cortical tubular epithelium and cystic tubules were frequently observed. Renal adenomas, occasionally multiple, were seen in 8/8 females and 3/7 males surviving to 21 months (Payne & Arena, 1978). Similar results were seen in dogs (Payne & Arena, The subacute and chronic toxicity of SK&F 36914 and SK&F D-39162 in dogs, 1978).
Szabo et al 1978a reported the effects of gold-containing compounds, including Auranofin® on pregnant rats and fetuses. The effects of gold sodium thiomalate and the oral gold compound Auranofin® on maternal and fetal toxicity and teratogenicity were investigated. Oral gold was administered by intubation on days 6-15 of pregnancy, while gold sodium thiomalate was administered on days 6-15 by subcutaneous injection. This was a standard exposure period in such studies and this exposure is considered to be equivalent to the first trimester of a human pregnancy. Standard procedures were used to examine fetuses and group sizes were adequate for the purpose of the study. Maternal and fetal toxicity was evident and fetuses of gold sodium thiomalate-dosed animals showed a pattern of dose-related malformations. The doses used led to death of a proportion of the dams and showed marked effects on body weight (including actual weight loss at the start of dosing) and reduced food consumption. The malformations included skeletal anomalies, external malformations and degrees of hydrocephalus and ocular defects. SK&F D-39162 did not affect food intake or weight gain, but was also associated with reductions in fetal weight compared to controls. The only major defect found with SK&F D-39162 treatment was edema. There was no evidence of an effect of gold sodium thiomalate on implantation, resorption, fetal number or fetal weight in the gold sodium thiomalate-treated animals. These authors concluded that the effects on the fetus were indirect and were attributable to accumulation of gold in the lysosomes of the visceral yolk sac epithelium, with consequent inhibition of vital enzymes involved in fetal nutrition. This hypothesis was advanced to explain the teratogenicity of other chemicals and could be plausible (Szabo, Guerriero, & Kang, The effects of gold containing compounds on pregnant rats and their fetuses, 1978).
Szabo et al 1978b reported the effects of gold-containing compounds on pregnant rabbits and fetuses. In this study pregnant rabbits were dosed from days 6-18 of pregnancy. Gold sodium thiomalate was administered by sub-cutaneous injection and oral compounds were given by intubation. Both routes of administration led to maternal deaths and abortions were also observed in surviving animals. Dose-related decreases in maternal food consumption, leading to actual body weight losses, were observed at the higher doses of both injected and oral gold. Effects were also evident on litter sizes, numbers of resorptions and mean fetal weights. Fetal anomalies and malformations were also observed, primarily in the abdomen (gastroschisis and umbilical hernia), with a lower incidence of anomalies affecting the brain, heart lungs and skeleton. The authors concluded that the incidence of abdominal anomalies, exceeding all of their historical control data, indicated a specific sensitivity in the rabbit to such an effect of gold (Szabo, DiFebbo, & Phelan, 1978).
Based on these studies, oral administration of relatively high doses of gold-containing compounds was associated with a dose-related incidence of erosions of the gastric mucosa and, in a longer duration study, of significant ileocaecal lesions (including ulceration) that caused the deaths of a number of animals. Examination of the data presented suggested that the gastric lesions were typical of a marked direct local effect on the mucosa. The renal cortical tubular epithelium was another target tissue, perhaps through the development of high local concentrations during the concentration of the urine. The cortical tubular epithelium lesions progressed from karyomegaly to adenoma formation in a significant number of animals. Although this is a benign tumor it cannot be ignored in terms of risk assessment. However, it is also notable that lesions of the rodent kidney are relatively common, particularly in males, but these appeared to affect females relatively more than males in these studies.
The gastric lesions occurred after administration of relatively large amounts of gold solutions. There was also a suggestion in these studies that the important toxic agent is ionic gold (e.g., Au (III) or Au3+). Lesions of this type are also produced by many NSAID agents used in the treatment of various forms of arthritis and are generally considered to be a manageable, albeit undesirable, side effect. Accordingly, the absence of such negative effects would constitute an advantage over existing gold-based therapies.
Cheriathundam and Alvares in 1996 evaluated the effects of sodium gold thiolate and Auranofin® on liver and kidney markers and metallothionein levels in the Sprague Dawley rat and three strains of mouse (Swiss-Webster, C3H/Hej and DBA/2J). In the rat, gold sodium thiolate led to a 7-fold increase in liver metallothionein levels, whereas in the mouse strains metallothionein levels increased 2-fold in the Swiss-Webster and about 5-fold in the inbred strains. Gold sodium thiolate led to only minimal changes in renal metallothionein levels in the mouse strains. The liver marker serum ALAT was not altered by gold sodium thiolate in any of the species or strains tested. BUN, an indicator of kidney function, was elevated 3-fold in rats but not in any of the mouse strains. These data are consistent with the observation that gold sodium thiolate is nephrotoxic in rats and humans, but it is interesting to note the lack of evidence of nephrotoxicity in the mouse (Cheriathundam & Alvares, 1996).
The observation of embryonic toxicity and fetal defects after treatment of pregnant animals of two species suggests the possibility that gold in many, if not all previously used forms, represents a developmental risk. This has parallels with many other current RA therapies, in which methotrexate, for example, is subject to label warnings regarding potential harmful effects on the fetus.
Several possible pharmacological actions contributing to both clinical efficacy and adverse reactions have been identified for oral gold. For example, Walz and his colleagues showed that Auranofin® inhibited carrageenan-induced edema in rats in a dose-related fashion in concentrations of 40, 20 and 10 mg/kg with maximum inhibition of 86% at the highest dose, and a serum gold level of approximately 10 μg/mL. The two basic ligands of Auranofin®, namely triethylphosphine oxide and 2,3,4,6-tetra-o-acetyl-1-thio-β-D glucopyranose did not show any significant biological activity, and gold sodium thiomalate, gold thioglucose and thiomalic acid did not significantly affect rat paw edema. Auranofin® was shown to significantly suppress adjuvant arthritis, whereas the ligands were without any effect. Auranofin® inhibited antibody dependant complement lysis. Auranofin® has been shown to inhibit the release of lysosomal enzymes such as β-glucuronidase and lysozyme from stimulated polymorphs. Auranofin® is a potent inhibitor of antibody dependent cellular cytotoxicity exhibited by polymorphs from adjuvant arthritic rats. Auranofin® is a much more potent inhibitor of superoxide production than gold sodium thiomalate. In an immune phagocytosis assay, gold sodium thiomalate showed no inhibitory activity at a concentration of 40 times that of Auranofin®, causing marked inhibition (Walz, DiMartino, Intocca, & Flanagan, 1983).
Walz and his colleagues also stated that Auranofin® was more potent than gold sodium thiomalate as an inhibitor of cutaneous migration, chemotaxis and phagocystosis by peripheral blood monocytes. Lipsky and his colleagues showed that Auranofin®, like gold sodium thiomalate, inhibited lymphoblastogenesis in vitro by directly inhibiting of mononuclear phagocytes. However, Auranofin® also had an inhibitory effect on lymphocyte function, not observed with gold sodium thiomalate. Inhibition of monocytes was achieved with concentrations of Auranofin® which were 10 to 20 fold lower than those of the gold sodium thiomalate (Walz, DiMartino, Intocca, & Flanagan, 1983).
In general, patients with active rheumatoid disease have a decreased capacity for either mitogen-stimulated lymphoblastogenesis or for lymphoblastogenesis induced by the mixed lymphocyte reaction. Although patients initially treated with gold sodium thiomalate first showed some suppression of mitogen-stimulated lymphoblastogenesis, those who eventually responded to the drug showed normal lymphocyte responsiveness in vitro. In contrast, within a few weeks of patients receiving Auranofin®, lymphocyte responsiveness was markedly inhibited. Thus, Auranofin® exhibits a powerful immunosuppressant effect in vitro at an order of magnitude less than the injectable gold compounds, most likely due to the major differences in the pharmacological properties of the oral compounds versus the injectable gold-thiol compounds (Dabrowiak, 2009).
Adverse reactions were the major limiting factor to the use of oral gold compounds such as Auranofin®, in that approximately 30-50% of treated patients developed some form of toxicity (Dabrowiak, 2009) (Kean & Anastassiades, 1979) (Kean & Kean, The clinical Pharmacology of Gold, 2008).
Skin rash was the most common negative side effect and some form of rash occurred in approximately 30% of patients. Most lesions occurred on the hands, forearm, trunk and shins, but occasionally occurred on the face and were slightly erythematosus with scaly patches, 1-10 cm in size, resembling a seborrheic rash. Severe problems of skin rash in the form of nummular eczema, total exfoliation and intense pruritus have been recorded as rare.
Oral ulcers (painful and pain free) resembling the aphthous ulcer, occurred in approximately 20% of patients who received injectable gold therapy. The development of a mouth ulcer was a definite contraindication to continuation of gold therapy since it was known that oral ulceration could herald pemphigold-like bullous skin lesions.
The frequency of proteinurea varied widely (0-40%) in the studies reported by Kean and Anastassiades, most likely reflecting different definitions as to what constitutes proteinurea. In these studies there are no well documented cases of any long term serious or permanent renal damage due to gold therapy; however microscopic haematuria was a cause for discontinuing oral gold treatment (Kean & Anastassiades, 1979).
Thrombocytopenia due to gold compounds occurred as two distinct types: the more usual was associated with platelet surface IgG antibody and the other less common was secondary to bone marrow suppression. The genetic marker HLA DR3 may indicate an increased risk of a patient developing thrombocytopenia associated with platelet surface antibodies.
Idiopathic toxicities in the form of cholestatic jaundice or acute enterocolitis have also been associated with the injectable gold compounds, particularly gold sodium thiomalate, but have not been reported with oral gold.
The deposition of elemental gold in the lens of the eye and the cornea has been reported, but this did not seem to result in any specific damage to visual acuity.
Specific to oral gold therapy was the development of loose soft stools, usually in the first month of therapy. The lower incidence of altered stools in later treatment months may be related to an earlier drop-out of those patients susceptible to the diarrhea. The development of frank watery diarrhea occurred in 2-5% of patients and appeared to be dose-related.
In general the adverse event incidence is lower with oral gold than injectable gold, but can still be substantial.
A second major drawback to the use of available gold-based treatments is the very slow onset of efficacy. Patients often must continue treatment with, for example, gold salts for three to six months before experiencing any significant benefit. This long wait for any observed benefit is a major impediment to patient compliance and therefore adversely affects efficacy in use.
The knowledge of the pharmacokinetic profiles of gold is largely centered on the measurement of the element Au, but not much is known of the gold structure (e.g., its chemical or physical or crystalline structure) when the gold is present in various tissues or organs.
After oral ingestion, oral gold complexes are rapidly, but incompletely, absorbed. The gold moiety of the injectable gold complex seems to be rapidly absorbed into the circulation after intramuscular injection. In blood circulation, Auranofin® (or ligands thereof) seem to be bound predominately to albumin. Specifically, after oral administration of radiolabeled Auranofin® to human volunteers, approximately 25% of the administered dose was detected in the blood plasma, with peak concentrations of 6-9 μg/100 mL being reached within 1-2 hours. The plasma half life was on the order of 15-25 days with almost total body elimination after 55-80 days. Only about 1% of radiolabeled Au was detectable after 180 days, whereas up to 30% of gold from gold sodium thiomalate was detected at this time. The gold was widely distributed throughout the reticulo-endothelial system, particularly in the phagocytic cells of the liver, bone marrow, lymph nodes, spleen, and also in the synovium. Deposition in the skin occurred and it has been observed that there may be a quantitative correlation between the amount of gold in the dermis and the total dose of gold given. Electron dense deposits of gold were also observed in the tubular cells of the kidney, another site rich in sulphydryl-containing enzymes, but the presence of gold associated with the glomerulus does not appear to be common (Walz, DiMartino, Intocca, & Flanagan, 1983) (Dabrowiak, 2009).
Gold Nanoparticles
Other formulations of gold have been and continue to be developed, most of which utilize gold nanoparticles made by a variety of chemical reduction techniques; and some of which utilize an underwater plasma arcing technique; and most of which result in various stable or partially stable gold colloids or gold nanoparticle suspensions.
Colloidal Gold Nanoparticles by Chemical Reduction
Michael Faraday made the first colloidal gold suspension by chemical reduction methods around the 1850's (Faraday, 1857). Faraday used reduction chemistry techniques to reduce chemically an aqueous gold salt, chloroaurate (i.e., a gold (III) salt), utilizing either phosphorous dispersed into ether (e.g., CH3—CH2—O—CH2—CH3), or carbon disulfide (i.e., C2), as the reductant.
Today, most colloidal gold preparations are made by a reduction of chloric acid (hydrogen tetrachloroaurate) with a reductant like sodium citrate to result in “Tyndall's purple.” There are now a variety of “typical” reduction chemistry methods used to form colloidal gold. Specifically, several classes of synthesis routes exist, each of which displays different characteristics in the final products (e.g., colloidal gold nanoparticles) produced thereby. It has been noted that in addition to the strength, amount and type of the reductant utilized, the action of a stabilizer (i.e., the chemical utilized in the solution phase synthesis process) is critical (Kimling, 2006).
While Faraday introduced colloidal gold solutions, the homogenous crystallization methods of Turkevich and Frens (and variations thereof) are most commonly used today and typically result in mostly spherical-shaped particles over a range of particle sizes (Kimling, 2006). Specifically, most current methods start with a gold (III) complex such as hydrogen tetrachloroaurate (or chloric acid) and reduce the gold in the gold complex to gold metal (i.e., gold (0) or metallic gold) by using added chemical species reductants, such as Na thiocyanate, White P, Na3 citrate & tannic acid, NaBH4, Citric Acid, Ethanol, Na ascorbate, Na3 citrate, Hexadecylaniline and others (Brown, 2008). However, another chemical reduction technique uses sodium borohydride as a chemical species reductant for AuP (Ph3) (Brown, 2008). Depending on the particular processing conditions utilized in these chemical reduction processes, the sizes of these mostly spherical nanoparticles formed range from about 1 nm to about 64 nm in diameter (Brown, 2008). Additionally, specific thermal citrate reduction methods utilized by Kimling resulted in a small fraction of triangular-shaped particles, in addition to spherical-shaped particles, with the triangular-shaped species at most being about 5% (Kimling 2006).
Additional work has focused on controlling shapes of colloidal metal nanoparticles. Biologists and biochemists have long understood that “structure dictates function” with regard to protein functioning. Gold nanoparticles of different shapes also possess different properties (e.g., optical, catalytic, biologic, etc.). Controlling nanoparticle shape provides an elegant approach to, for example, tune nanoparticles optically. While all gold nanoparticles contain a lattice that is face-centered cubic, if permitted or caused by certain processing conditions, gold nanoparticles can adopt a variety of crystalline shapes ranging from irregular ellipsoids with defect loaded surfaces (e.g., steps) to polyhedra with comparatively limited surface defects. Different crystalline morphologies are associated with different crystal planes (or sets of crystal planes). However, some of the most common gold nanoparticle morphologies are not composed of single domains, but rather are made of twinned planes (Tao, 2008).
Yuan, et al. recognized that non-spherical-shaped gold nanoparticles could be most readily achieved by providing seed crystals from a borohydride reduction of a gold salt (i.e., HAuCl4 or auric acid). The seed crystals were then placed into contact with the same gold salt in solution with the chemical species NH2OH, CTAB and sodium citrate being added as reductants and/or surfactants (e.g., capping agents). Several different crystalline shapes were formed by this approach including triangular, truncated triangular, hexagonal layers and pseudo-pentagonal. Yuan concluded that variations in processing by using different chemical reduction techniques can influence the physical and chemical properties of the resulting particles. The researchers noted that the choice of a capping agent was a key factor in controlling the growth (and shape) of the nanoparticles (Yuan, 2003).
The process described and used by Yuan is known as “heterogeneous nucleation” where seed particles are produced in a separate synthetic step. Thus, this type of shape control can be considered an overgrowth process (Tao, 2008). Many chemical reduction techniques utilize this more complex two-step heterogeneous nucleation and growth process. However, others use a single step homogenous nucleation whereby seed crystals are first nucleated and nanoparticles are then formed from the nucleated seed crystals. Typically, a series of chemical reactions occur simultaneously in homogeneous nucleation. A main goal in homogenous nucleation is to balance the rate of nucleation against the rate of crystal growth and to control particle size because both nucleation and growth proceed by the same chemical process(es) (Tao, 2008).
Metal nanoparticle synthesis in solution(s) commonly requires the use of surface-active agents (surfactants) and/or amphiphilic polymers as stabilizing agents and/or capping agents. It is well known that surfactants and/or amphiphilic polymers serve critical roles for controlling the size, shape and stability of dispersed particles (Sakai, 2008).
Some of the most common crystal morphologies observed in crystalline gold nanoparticles (for example in heterogeneous nucleation processes) do not consist of single crystals or single domains, but rather particles containing multiple crystal domains, often bounded by twin planes. A regular decahedron (also referred to as a pentagonal bi-pyramid) is an equilibrium shape bound completely by triangular (III) facets and can be thought of as five tetrahedral sharing a common edge along a fivefold axis. These structures are commonly observed for nano-crystalline particles synthesized by metal evaporation onto solid substrates and seeded heterogeneous nucleation reduction chemistry approaches (Tao, 2008). However, for nanoparticles synthesized by the methods of Turkevich and Frens, decahedra are difficult to observe because they function as favorable seeds for the growth of nanowires and nanorods (Tao, 2008). Thus, a variety of shapes can be achieved by controlling processing conditions, along with the amounts and types of surfactants and capping agents added and used during the reduction chemistry approaches attributed to Turkevich and Frens.
In each of the colloidal gold compositions produced by reduction chemistry approaches, it is apparent that a surface coating comprising one or more elements of the reductant and/or the surfactant or capping agent will be present on (or in) at least a portion of the suspended gold nanoparticles. The use of a reductant (i.e., a reducing agent) typically assists in suspending the nanoparticles in the liquid (e.g., water). However, the reducing agent coating or surface impurity is sometimes added to or even replaced by surfactant coatings or capping agents. Such reductant/surfactant coatings or films can be viewed as impurities located on and/or in the metal-based nanoparticles and may result in such colloids or sols actually possessing more of the properties of the protective coating or film than the gold nanoparticle per se (Weiser, p. 42, 1933).
For example, surfactants and amphiphilic polymers become heavily involved not only in the formation of nanoparticles (thus affecting size and shape), but also in the nanoparticles per se. Surface properties of the nanoparticles are modified by reductant coatings and/or surfactant molecule coatings (Sperling, 2008).
Absorption of a hydrophobic tail, a hydrophilic head group and certain counter ions (at least in the case of the use of ionic surfactants) on the surface of nucleated particles, as well as complexation of metal ions with surfactants and/or amphiphilic polymers with the formed particles, all can influence the shape of the nanoparticles, the surface of the nanoparticles and/or alter the functioning of the nanoparticles (Sakai, 2008).
Different surface chemistries or surface films (e.g., the presence of reductant by-product compositions and/or thicknesses (e.g., films) of reductant by-products) can result in different interactions of the gold nanoparticles with, for example, a variety of proteins in an organism. Biophysical binding forces (e.g., electrostatic, hydrophobic, hydrogen binding, van der Waals) of nanoparticles to proteins are a function not only of the size, shape and composition of the nanoparticles, but also the type of and/or thickness of the surface impurities or coating(s) on the nanoparticles. The Turkevich and Frens methods (and variations thereof) for making gold nanoparticles are the most widely understood and utilized chemical reduction processes. The use of a citric acid or sodium citrate reductant results in citrate-based chemistries (e.g., a citrate-based coating) on the surface of the gold nanoparticle (i.e., also referred to as citrate-stabilized) (Lacerda, 2010).
Further, Daniel et al. reviewed the major gold nanoparticle formation techniques, including the chemical synthesis and assembly processes including: (1) citrate reduction, which results in “a rather loose shell of [citrate-based] ligands” attached to the gold nanoparticles; (2) a variation of the citrate reduction method which uses a citrate salt and an amphiphile surfactant (for size control); (3) the “Brust-Schiffrin” methods which result in thiol or thiolate ligands “that strongly bind gold”; (4) methods that result in sulfur-containing ligands including xanthates, disulfides, dithiols, trithiols and resorcinarene tetrathiols; and (5) other ligands that relate to phosphine, phosphine oxide, amines, carboxylates, aryl isocyanides, and iodides (which can replace citrate coatings). The authors reiterated statements attributed to Brust regarding formed gold nanoparticles: “The resulting physical properties are neither those of the bulk metal nor those of the molecular compounds, but they strongly depend on the particle size, interparticle distance, nature of the protecting organic shell, and shape of the nanoparticles.” (Daniel, 2004)
While the organic ligands present on the gold nanoparticles (e.g., citrate-based ligands or coatings or films) helps to stabilize the gold nanoparticles in the liquid to prevent the nanoparticle from, for example, being attached to other nanoparticles and agglomerating and/or settling out of suspension due to, for example, gravity, these organic-based ligands (e.g., organic shells) are impurities (i.e., relative to the underlying gold nanoparticle) and contribute to the gold nanoparticle's interaction with proteins in a living system. Such coating(s) or film(s) can have strong biological influences (Lacerda, 2010).
Further, Wang et al concluded that the commonly used citrate-reduced gold nanoparticles interfere with the uptake of gold nanoparticles relative to reductant and stabilizer-free colloidal solutions (Wang, 2007).
Likewise, Lacerda, et al. stated that a better understanding of the biological effects of nanoparticles requires an understanding of the binding properties of the in-vivo proteins that associate themselves with the nanoparticles. Protein absorption (or a protein corona) on nanoparticles can change as a function of nanoparticle size and surface layer composition and thickness. Lacerda concluded that the protein layers that “dress” the nanoparticle control the propensity of the nanoparticles to aggregate and strongly influence their interaction with biological materials (Lacerda, 2010).
Cleaning Colloidal Gold Nanoparticles Made by Chemical Reduction Techniques
In some cases, the reductant surface coating or film is permitted to remain as an impurity on the surface of the nanoparticles, but in other cases, it is attempted to be removed by a variety of somewhat complex and costly techniques. When removed, the coating typically is replaced by an alternative composition or coating to permit the nanoparticles to stay in suspension when hydrated. The influence of purity on the chemistry and properties of nanoparticles is often overlooked; however, results now indicate that the extent of purification can have a significant impact (Sweeney, 2006). These researchers noted that sufficient purification of nanoparticles can be more challenging that the preparation itself, usually involving tedious, time-consuming and wasteful procedures such as extensive solvent washes and fractional crystallization. Absent such purification, the variables of surface chemistry-related contaminants on the surface of chemically reduced nanoparticles affects the ability to understand/control basic structure-function relationships (Sweeney, 2006).
Subsequent processing techniques may also require a set of washing steps, certain concentrating or centrifuging steps, and/or subsequent chemical reaction coating steps, all of which are required to achieve desirable results and certain performance characteristics (e.g., stabilization due to ligand exchange, efficacy, etc.) for the nanoparticles and nanoparticle suspensions (Sperling, 2008). In other cases, harsh stripping methods are used to ensure very clean nanoparticle surfaces (Panyala, 2009).
Thus, others have concluded that the development of gold nanoparticles in the management, treatment and/or prevention of diseases is hampered by the fact that current manufacturing methods for gold nanoparticles are by-and-large based on chemical reduction processes. Specifically, Robyn Whyman, in 1996, recognized that one of the main hindrances in the progress of colloidal golds manufactured by a variety of reduction chemistry techniques was the lack of any “relatively simple, reproducible and generally applicable synthetic procedures” (Whyman 1996). There are many variations of the original reduction chemistry techniques taught by Faraday each of which can produce colloidal gold having a variety of different physical properties (e.g., alone or in suspension) and reductant coatings, all of which can result in different efficacy/toxicity profiles when used in or with living cells. None of these techniques meet Whyman's criteria. Accordingly, a relatively simple, reproducible and generally applicable manufacturing approach for making gold nanocrystals would be welcomed. Further, the ability of such a manufacturing approach to be compliant with FDA cGMP requirements would be even more valuable.
Others have begun to recognize the inability to extricate completely adverse physical/biological performance of the formed nanoparticles from the chemical formation (i.e., chemical reduction) processes used to make them. In this regard, even though somewhat complex, expensive and non-environmentally friendly, washing or cleaning processes can be utilized to alter or clean the surface of nanoparticles produced by reduction chemistry, elements of the chemical process may remain and affect the surface of nanoparticles (and thus their functioning). Moreover, the presence of certain chemicals during the nanoparticle formation process affects the morphology (i.e., size and/or shape) of the forming nanoparticles. Certain possible desirable morphologies (shapes) known to exist in gold-based crystalline systems are not readily observed in many products produced by these reduction chemistry techniques.
Other Techniques for Making Colloidal Gold
Obtaining a surfactant and reducer-free (e.g., no stabilizing, capping or reducing agents added to achieve reduction of gold ionic species) has become a goal of certain researchers who apparently understand some adverse consequences of reductant/surfactant coatings being present from reduction chemistry approaches. For example, ultrasound techniques have been used whereby a 950 kHz frequency is applied to an aqueous hydrogen tetrachloroaurate solution. Spherical gold nanoparticles in the range of 20-60 nm were prepared at temperatures above 50° C., while relatively larger triangular plates and some hexagonal spheres coexisted when the mixture was processed below 50° C. (Sakai, 2008).
X-ray irradiation of HAuCl4 has been developed to obtain reductant and stabilizer-free gold nanoparticles so as not to “jeopardize” biocompatibility issues in biomedical applications. The authors speculated that they generated the required electrons for chemical reduction of Au+ by using “intense” X-ray beams to create a hydrogen-free radical electron donor (Wang, 2007).
Another older and more complex technique for minimizing or eliminating the need for reducing agents and/or minimizing undesirable oxidation products of the reductant utilizes γ-irradiation from a 60Co source at a dose rate of 1.8×104 rad/h. In this instance, Au (CN)2 was reduced by first creating hydrated electrons from the radiolysis of water and utilizing the hydrated electrons to reduce the gold ions, namely:eaq−+Au(CN)2→Au0+2CN−  (Henglein, 1998).
It is known that the surface of the gold nanoparticle may be further processed by adding chemical species, such as polyehteylene glycol (PEG), or other specific ligands. In this regard, extensive work has occurred in therapies for cancer where PEG-coated gold nanoparticles are induced by a variety of techniques to migrate to a cancer or tumor site and are thereafter irradiated with, for example, infrared or radiowaves to heat and destroy cancer cells (Panyala, 2009). Surface PEGylation is also known to increase the blood half-life of nanoparticles; and polysorbate-80 can improve the blood-brain-barrier transport of nanoparticles (Teixido & Giralt, 2008).
Colloidal Gold by Underwater Arcing
Also known in the art are methods for making gold nanoparticles by an underwater arcing method. This method was first pioneered by Bredig in the late 1800's. Bredig used a direct current to create an underwater arc between two wires. Bredig used a current of 5-10 amps and a voltage of 30-110 volts. In some cases, Bredig also used 0.001N sodium hydroxide instead of pure water. Bredig thought of his process as pulverizing the metallic electrodes. Bredig obtained hydrosols of gold in this manner (Weiser, pp. 9-17, 45-46, 1933).
Svedberg later improved on Bredig's process by utilizing a high frequency arc instead of the direct current arc of Bredig. Svedberg pointed out that the arc permits the formation of a metal gas which subsequently condenses into particles of colloidal dimensions. Much debate surrounded the exact mechanisms of the process, however vaporization of the metal was viewed as being important (Weiser, pp. 9-17, 45-46, 1933).
The parameters of greatest interest to Svedberg in controlling the electric pulverization process to form colloid solutions were, a) the rate of pulverization, b) the ratio of sediment to total metal dispersed, c) the extent of decomposition of the medium, and d) the dependence of (a)-(c) on the current characteristics. The amount of sediment achieved by the Bredig and Svedberg processes ranged from about 30% to about 50%, under a variety of processing conditions (Kraemer, 1924).
More recent work with the Bredig process on palladium was performed by Mucalo, et al. These investigators tested the theory of whether the metallic particles in Bredig sols were “impure” due to impurities from the concurrent electrolyte decomposition of the electrolyte and oxidized material thought to form during arcing (Mucalo, 2001). These investigators utilized modern surface analytical techniques (i.e., XPS, or “x-ray photoelectron spectroscopy”) to determine differences in surface speciation as a function of pH. At lower pH's a grey-black unstable material was produced. At higher pH's, the sol was more stable, but still completely aggregated within 1-2 weeks. Nanoparticles produced consisted of irregularly-shaped spheres. While materials produced at both higher and lower pH's were mostly metallic in character, the surface characteristics of these unstable colloids were different. The higher pH Bredig sols resulted in a thicker outer oxide layer on the unstable nanoparticles (Mucalo, 2001).
The methods of Bredig and Svedberg were subsequently improved on by others to result in a variety of underwater arc-based methods. However, common to each of these underwater arcing methods is the result of somewhat irregularly-shaped metallic-based spheres. In this regard, the nanoparticles produced by the Bredig or Svedberg processes are non-specific, spherical-like shapes, indicative of a metal-based vaporization followed by rapid quench methods, the nanoparticles being coated with (and/or containing) varying amounts of different oxide-based materials.
Toxicology of Colloidal Gold Nanoparticles
A review on the toxicology of gold nanoparticles was performed by Johnston, et al. and reported in 2010. There were four intravenous exposure routes summarized for both mice and rats and an intratracheal approach for rats. Regarding the four intravenous studies summarized, Johnston, et al. reported that tissue sites of accumulation, in order of quantity were liver-spleen in 3 of 4 tests and liver-lung in 1 of 4 tests (i.e., highest gold nanoparticle accumulation was in the liver). Specifically, the four intravenous tests reported by Johnston et al. are summarized below (Johnston, 2010).
The tissue distribution of metal particles, following exposure via a variety of routes (Johnston, et al., 2010).
Tissue sites ofaccumulation SizeExposure(in order ofPaperNP(nm)Routequantity)ConclusionCho, et al., 2009Gold13IntravenousLiver, spleen,The primary sites of(PEG(mice)kidney, lung,accumulation are thecoated)brainliver and spleen, NP'saccumulate withinmacrophagesDe Jong, et al.,Gold10, 50,IntravenousLiver, spleen,Wider organ distribution2008100,(rats)lungs, kidneys,of smaller particles,250heart, brain,whereas larger particlesthymus, testiswere restricted to theliver and spleenSemmler-Gold4 andIntravenousLiver, spleen,Small particlesBehnke, et al.,18(rats)kidneys, skin,demonstrate a more2008GIT, heartwidespreadaccumulation/distributionSonavane, et al., Gold15, 50,IntravenousLiver, lung,Wider tissue distribution2008b100,(mice)kidneys, spleen,for smaller particles-200brain15- and 50-nm NP'saccumulated within the
Johnston, et al. were critical of a variety of uncertainties introduced into a number of the reviewed toxicology studies including that certain conclusions (made by others) regarding toxicity as a function of only particle size were not accurate. Specifically, Johnston, et al. reported that Pan et al (in 2007) concluded that 1.4 nm gold nanoparticles were the most toxic gold nanoparticles tested out of a range of nanoparticle sizes, including 1.2 nm diameter gold nanoparticles. While Pan, et al. believed there to be a difference in toxicity profile as a function of size, Johnston, et al. noted that the 1.4 nm particles were made by the investigators themselves and the 1.2 nm particles were obtained from an outside company (thus suggesting that there were different surface characteristics of both nanoparticles). Johnston, et al. concluded that “agglomerations states or surface chemistry” were the reason(s) for differential performance with both being “known to alter particle behaviour and toxicity” (Johnston, 2010).
Johnston, et al. also concluded that experimental setup influences toxicity results; and that the tissue distribution of gold nanoparticles in an organism is a function of the exposure route, as well as size, shape and surface chemistry of nanoparticles. Additionally, they observed that the liver appears to be the primary site of accumulation and speculated that result is due to the presence of macrophages in the liver. They also noted that nanoparticle uptake is probably a result of the type and extent of protein binding occurring on the surface of the nanoparticles (e.g., a protein corona) which is a function of the size, shape and surface coating on the nanoparticles. In particular, they noted that the ability of a variety of cell types to internalize nanoparticles by, for example, endocytosis. This endocytosis mechanism which appeared to be a function of particle shape, as well as particle surface characteristics, such as protein absorption on the surface thereof. In other words, biological uptake is a function of shape, size and charge; and is also very serum-dependent (Johnston, 2010).
Efficacy of Colloidal Gold
Work by Abraham and Himmel (reported in 1997) disclosed the use of colloidal gold in the treatment of 10 patients who previously did not respond to a variety of other gold-based treatments. The colloidal gold used in the study was made by a variation of the standard “citrate method” of Maclagan and Frens with “several proprietary modifications.” Maltodextrins (Food Grade) were used at a concentration of 2.5% to prevent autoaggregation of the gold particles (Abraham, 2008). The sizes of the colloid particles produced were reported as being less than 20 nm, as confirmed by a process of passing the colloidal suspension through a 20 nm filter (i.e., produced by Whatman Anotop). Subsequent TEM work caused Abraham to conclude that 99% of the particles produced were less than 10 nm. Sodium benxoate was also added (Abraham, 2008).
The colloidal gold suspension resulted in a 1,000 mg/L (i.e., 1,000 pm) concentration. The dosage level provided to each patient varied between 30 mg/day and 60 mg/day, with most dosages being 30 mg/day, for a 24 week period. These dosages were taken orally. Table 1 therein lists the patient's sex, age and previous conditions and/or treatments. The article concludes that 9 of the 10 patients “improved markedly by 24 weeks of intervention” (Abraham & Himmel, 1997). Abraham also reported a lowering of certain cytokine concentrations including IL-6 and TNF (Abraham, 2008).
Work on collagen-induced arthritis in rats by Tsai concluded that nanogold particles bound to the protein VEGF and that such binding was the reason for an improved clinical performance of rats that were intra-articularly injected with colloidal gold. In this case, the injected colloidal gold was prepared by the standard chemical reduction method of utilizing a gold chloroaurate reduced with sodium citrate. Tsai, et al. reported that the gold nanoparticles were spherical having an approximate diameter of 13 nm, as measured by transmission electron microscopy. The concentration of the intra-articular solution was 180 μg/ml (i.e., 180 ppm). The intra-articular injection was made one time, either on day 7 or day 10 after induction of CIA (Tsai, 2007).
Brown, et al. disclosed in 2007 that a standard colloidal gold preparation (referred to as Tyndall's purple) was prepared by standard chemical reduction methods, namely, the reduction of chloroauric acid with sodium citrate. The average particle size of the gold nanoparticles produced was 27+/−3 nm. This colloidal gold was dispersed in isotonic sorbitol and injected by a parenteral and subcutaneous approach into rats that experienced experimentally induced arthritis. The dose injected was at a concentration of 3.3 μg/kg. Brown, et al. also disclosed that colloidal gold, when administered subcutaneously, was approximately 1,000 times more effective than the comparative sodium aurothiomalate. Brown, et al. also disclosed that the colloidal gold was ineffective when given orally and concluded that the ineffectiveness was due to coagulation of the gold nanoparticles in the presence of gastric juice and sodium chloride (Brown, 2007).
Brown, et al. reviewed alternative preparation methods for colloidal gold having a variety of sizes and shapes (Brown, 2008). Brown, et al. disclosed in Table 2 a variety of properties associated with “nano-gold hydrosol.” The authors concluded that the studies conducted by them (and reviewed by them) “suggest that gold nanoparticle (Au0)-based drugs may play a role in future clinical therapies targeted to regulating macrophages” (Brown, 2008).
The references cited throughout the “Background of the Invention” are listed below in detail.    Abraham, G. E. & Himmel, P. B. (1997). Management of rheumatoid arthritis: rationale for the use of colloidal metallic gold. J. Nutr. Environ Med. 7, 295-305.    Abraham, G. E. (2008). Clinical Applications of Gold and Silver Nanocolloids. Original Internist, 132-157.    Agata, N., et al. (2000). Suppression of type II collagen-induced arthritis by a new Isocoumarin, NM-3. Res Commun Mol Pathol Pharmacol., 108 (5-6), 297-309.    Brown, C. L., Whitehouse, M. W., Tiekink, E. R. T., & Bushell G. R. (2008). Colloidal metallic gold is not bio-inert. Inflammopharmacology, 16, 133-137.    Brown, C. L., et al. (2007). Nanogold-pharmaceutics (i) The use of colloidal gold to treat experimentally-induced arthritis in rat models; (ii) Characterization of the gold in Swarna bhasma, a microparticulate used in traditional Indian medicine. Gold Bulletin, 2007, 40 (3), 245-250.    Cheriathundam, E., & Alvares, A. (1996). Species differences in the renal toxicity of the antiarthritic drug, gold sodium thiomalate. J Biochem Tox, 11(4), 175-81.    Dabrowiak, J. (2009). Gold Complexes for Treating Arthritis Cancer and Other Diseases. In J. Dabrowiak, Metals in Medicine (pp. 191-217). Chichester UK: John Wiley and Sons.    Daniel, M. C. & Astruc, D. (2004). Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev., 104, 293-346.    Eisler, Ronald. Biochemical, Health, and Ecotoxicological Perspectives on Gold and Gold Mining. Boca Raton: CRC Press, 2004.    Faraday, M. (1857). The Bakerian lecture: Experimental relations of gold (and other metals) to light. Philosoph. Trans. R. Soc. London, 147, 145-181.    Henglein, A. & Meisel, D. (1998). Radiolytic Control of the Size of Colloidal Gold Nanoparticles. Langmuir, 14, 7392-7396.    Ho, S., & Tiekink, E. (2005). Gold beased metalotherapeutics; Use and Potential. In M. Gielen, & E. Tiekink, Metallotherapeutic Drugs and Metal-Based Diagnostic Agents (pp. 507-527). Chictester: JH Wiley and Sons.    Johnston, H. J., Hutchinson, G., Christensen, F. M., Peters, S., Hankin, S. & Stone, V. (2010). A review of the in vivo and in vitro toxicity of silver and gold particulates: Particle attributes and biological mechanisms responsible for the observed toxicity. Critical Reviews in Toxicology, 40 (4), 328-346.    Kean, W., & Anastassiades, T. (1979). Long term chrysotherapy; incidence of toxicity and efficacy during sequential time periods. Arthritis Rheum, 22(5), 495-501.    Kean, W., & Kean, I. (2008). The clinical Pharmacology of Gold. Immunopharmacology, 16(3), 112-25.    Kimling, J., Maier, M., Okenve, B., Kotaidis, V., Ballot, H. & Plech, A. (2006). Turkevich Method for Gold Nanoparticle Synthesis Revisited. J. Phys. Chem. B, 110, 15700-15707.    Kraemer, E. O. & Svedberg, T. (1924). Formation of Colloid Solutions by Electrical Pulverization in the High-Frequency Alternating Current Arc. Journal of the American Chemical Society, 46 (9), 1980-1991.    Leonard, T. B., Graichen, M. E., Dahm, L. J., & Dent, J. G. (1986). Effects of the Chryosotherapeutic Agents Auranofin and Gold Sodium Thiomalate on Hepatic and Renal Drug Metabolism and Heme Metabolism. Biochemical Pharmacology, 35, (18), 3057-3063.    Mucalo, M. R. & Bullen, C. R. (2001). Electric arc generated (Bredig) palladium nanoparticles: Surface analysis by X-ray photoelectron spectroscopy for samples prepared at different pH. Journal of Materials Science Letters, 20, 1853-1856.    Panyala, N. G., Pena-Mendez, E. M., & Havel, J. (2009). Gold and nano-gold in medicine: overview, toxicology and perspectives. Journal of Applied Biomedicine, 7, 75-91.    Payne, B., & Arena, E. (1978). The subacute and chronic toxicity of SK&F 36914 and SK&F D-39162 in dogs. Vet Path, Suppl 5, 9-12.    Payne, B., & Arena, E. (1978). The subacute and chronic toxicity of SK&F 36914, SK&F D-39162 and gold sodium thiomalate in rats. Vet Path Suppl, 15(5), 13-22.    Sadler, P. J. (1976). The biological chemistry of gold: a metallo-drug and heavy-atom label with variable valency, Structure Bonding, 29, 171-215.    Shaw, C. F., III. (1999a). Gold complexes with anti-arthritic, anti-tumour and anti-HIV activity, in Uses of Inorganic Chemistry in Medicine, N. C. Farrell, (Ed.), Royal Society of Chemistry, Cambridge, UK, 26-57.    Shaw, C. F., III. (1999b). The biochemistry of gold, in Gold: Progress in Chemistry, Biochemistry and Technology, H. Schmidbaur, (Ed.), John Wiley & Sons, New York, 260-308.    Sakai, T., Enomoto, H., Torigoe, K., Kakai, H. & Abe, M. (2008). Surfactant- and reducer-free synthesis of gold nanoparticles in aqueous solutions. Colloids and Surface A: Physiocochemical and Engineering Aspects, 18-26.    Sperling, R. A., Gil, P. R., Zhang, F., Zanella, M., & Parak, W. J. (2008). Biological applications of gold nanoparticles. Chem. Soc. Rev, 37, 1896-1908.    Sweeney, S. F., Woehrle, G. H. & Hutchison, J. E. (2006). Rapid Purification and Size Separation of Gold Nanoparticles via Diafiltration. J. Am. Chem. Soc., 128, 3190-3197.    Szabo, K., DiFebbo, M., & Phelan, D. (1978). The effects of gold-containing compounds on pregnant rabbits and their fetuses. Vet Path, Suppl 5, 95-105.    Szabo, K., Guerriero, F., & Kang, Y. (1978). The effects of gold containing compounds on pregnant rats and their fetuses. Vet Path, 5, 89-86.    Tao, A. R., Habas, S. & Yang Peidong. (2008). Shape Control of Colloid Metal Nanocrystals. Small, 4 (3), 310-325.    Teixido, M. & Giralt, E. (2008). The role of peptides in blood-brain barrier nanotechnology. J. Pept. Sci., 14, 163-173.    Tsai, C., Shiau, A., Chen, S., Chen, Y., Cheng, P., Chang, M., et al. (2007). Amelioration of collagen-induced arthritis in rats by nanogold. Arthritis Rheum, 56(2), 544-54.    Ueda, S. (1998). Nephrotoxicity of gold salts, D-penicillamine, and allopurinol, in Clinical Nephrotoxins: Renal Injury from Drugs and Chemicals, M. E. De Broe, G. A. Porter, W. M. Bennett, and G. A. Verpooten, (eds.), Kluwer Dordrecht, 223-238.    USFDA (2005). Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Pharmacology and Toxicology.     Walz, D., DiMartino, M., Intocca, A., & Flanagan, T. (1983). Biologic actions and pharmacokinetic studies of Auranofin®. Am J Med, 759(6A).    Wang, C. H., et al. (2007). Aqueous gold nanosols stabilized by electrostatic protection generated by X-ray irradiation assisted radical reduction. Materials Chemistry and Physics, 106, 323-329.    Weiser, H. B. Inorganic Colloid Chemistry-Volume I: The Colloidal Elements. New York: John Wiley & Sons, Inc., 1933.    Whyman, R. (1996). Gold Nanoparticles A Renaissance in Gold Chemistry. Gold Bulletin, 29(1), 11-15.    Yuan, H., Cai, R. X. & Pang, D. W. (2003). A Simple Approach to Control the Growth of Non-spherical Gold Nanoparticles. Chinese Chemical Letters, 14 (11), 1163-1166.